SRA

We performed RNA-Seq and m6A RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-Seq) to determine whether the altered gene expression and the regulatory functions in cancer immunotherapy could be a consequence of Alkbh5 or Fto-mediated m6A/m6Am demethylation. We used two m6A peak calling algorithms to find the common peaks between the two methods, and only kept the conserved peaks detected in all the biological replicates of each group to obtain the most stringent results. Our data suggest that the enzymatic activity and the altered m6A/m6Am peaks of Fto and Alkbh5 knockout had similarity but also had obvious distinct features, which may lead to the different mechanisms of the two protein to exert their functions in regulating immunotherapy. In addition, we performed single cell RNA-seq on a tumor obtained from a patient who had stage IV melanoma progressive disease and responded well to PD-1 blockage therapy. We observed substantial immune cell infiltration and very few residual melanoma cells, indicators of good immunotherapy responses. We then examined ALKBH5 expression and found that there were more melanoma cells expressing ALKBH5 than the surrounding normal control cells. Overall design: NTC control, Alkbh5 KO, Fto KO mouse tumors after cancer immunotherapy were analyzed by RNA-Seq and MeRIP-Seq. Tumors from a melanoma patient who had stage IV melanoma progressive disease and responded well to PD-1 blockage therapy were analyzed by single cell RNA-seq.